Automated histology laboratory instrumentation has significantly improved the ability of pathology laboratories to process tissue samples, particularly biopsy samples, in a relatively rapid and consistent manner. These efforts have also reduced somewhat the dependence on skilled histology personnel and improved the quality of diagnostic material. Similarly, with all its limitations, the current evolution of slide-scanning technology has begun to make remote viewing and digital storage of tissue samples a reality. But there are aspects of traditional paraffin-embedded, microtome-cut, hematoxylin-eosin (H&E)-stained slices for routine pathologic evaluation that limit the ability to make more significant advances in the speed, quality, and completeness of tissue biopsy evaluation.
Visual examination of tissue samples remains a mainstay of diagnostic analysis of tissue but there is an increasing role of ancillary studies such as that derived from genetic and proteomic data. This trend is dependent on the availability of sufficient and adequately preserved tissue which competes with the interest for smaller samples and faster results. In addition, incomplete sample evaluation, artifacts of preparation, non-quantitative interpretation, limited growth pattern information, and an extended manual preparative process are some of the aspects of traditional slide-based histologic analysis of human samples that limit advancements in pathology. These are particularly relevant for the usual initial diagnostic step in pathologic assessment which is often core biopsies or fine needle aspirations.
Many alternative tissue processing and imaging approaches have been proposed to address limitations of traditional processing techniques. More recent ones include high-resolution x-ray computed tomography (Zehbe et al., 2010, J. R. Soc. Interface 7:49-59; Ritman, 2011, Annu. Rev. Biomed. Eng. 13:531-552) and optical coherence tomography (Zysk et al., 2007, J. Biomed. Opt. 12:051403-051403-21; Bizheva et al., 2005, J. Biomed. Opt. 10:11006-11006-07). These approaches have the advantages of being applicable to unprocessed fresh tissue and allowing complete 3-dimensional visual examination while leaving tissue unaltered and amenable to further characterization. At the present time, neither technique is able to produce images of sufficient resolution and contrast for adequate routine pathology evaluation.
Multiphoton microscopy (MPM), on the other hand, has the ability to provide images with excellent cellular detail and is a popular, powerful method for analysis of research samples. Use of short-pulse laser light also permits concurrent mapping of second-harmonic generation (SHG), making it possible to simultaneously produce quantifiable images of repeating asymmetric protein structures such as collagen and amyloid. Unfortunately, although the long wavelengths used in MPM can image deeper into tissue than confocal microscopy, traditional methods can only achieve clear images at depths of at most 50 μm with formalin-fixed specimens. Previous attempts to use MPM for imaging through fixed tissue have used serial sectioning (Ragan et al., 2007, J. Biomed. Opt. 12:014015-014015-9) or serial tissue ablation (Dechet et al., 1999, J. Urol. 162:1282-1284), both of which result in the destruction of the tissue specimen during the course of imaging, making them nonviable for routine clinical use.
The above noted points indicate that novel methods of tissue processing for imaging of uncut and un-embedded samples are desirable. Tissue clearing presents a useful approach to practically and significantly increase the accessible depth of imaging for various modes of optical sectioning microscopy. Past efforts to obtain high resolution images at depth with clearing have been limited to a small set of applications. These past approaches have failed to develop a processing method that can achieve the speed necessary for adequate implementation in routine pathology and many types of investigative work. They have also not been able to faithfully reproduce the types of coloration that trained specialists in morphologic evaluation are accustomed to interpreting.
Thus, there remains a need for a practical new processing method that can obtain high resolution images of tissue at depth in a relatively short period of time. Additionally, there is a need for these depth images to be obtained in a manner that makes them instantly recognizable by pathologists and microanatomy investigators. The present invention addresses this unmet need.